A characteristic polypeptide of Hailong baojia and its application

Abstract

The present invention relates to a characteristic polypeptide of the Baojia lance-mouthed sea dragon and its application and a method for identifying the Baojia lance-mouthed sea dragon, belonging to the field of biotechnology detection technology. The present invention provides a characteristic polypeptide unique to the Baojia lance-mouthed sea dragon, and the amino acid sequence of the characteristic polypeptide is shown in SEQ ID NO.1. The characteristic polypeptide provided by the present invention has excellent specificity and stability for the Baojia lance-mouthed sea dragon, has strong specificity, can be used for the identification of the Baojia lance-mouthed sea dragon, and has good application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of biotechnology detection, and in particular to a characteristic polypeptide of a sea dragon having a nose and nose, an application thereof, and a method for identifying a sea dragon having a nose and nose. Background Art

[0002] Sea dragon is an important animal medicinal material in my country. It has the effects of warming the kidney and strengthening yang, dispersing nodules and reducing swelling. It has a long history of use and many sources. The Chinese Pharmacopoeia stipulates that the medicinal source of sea dragon is the dried body of the Syngnathidae animals Solenognathus hardwickii (Gray), Syngnathoides biaculeatus (Bloch) or Syngnathusa cusLinnaeus.

[0003] As a tonic medicinal material, the market demand for sea dragons is increasing year by year. Since sea dragons have not been effectively cultivated artificially, the wild resources of sea dragons are decreasing day by day, and they are often confused with other varieties or sold as inferior products. At present, the sales proportion of sea dragons from different sources that are not included in the Chinese Pharmacopoeia is increasing year by year, resulting in chaos in the circulation of sea dragon medicinal materials. Baojia Gunwen Sea Dragon is one of the common mixed varieties on the market. As an individual completed sea dragon medicinal material, Baojia Gunwen Sea Dragon has relatively obvious characteristics for distinguishing properties. Once it is powdered or processed into an extract, the characteristics disappear and cannot be distinguished.

[0004] At present, the main methods for the identification and quality control of sea dragon species are trait identification, HPLC fingerprinting and molecular biology. The trait identification method generally requires that the sea dragon individual is complete and the appraiser has rich experience. This method has a certain degree of subjectivity; the HPLC fingerprint method has low specificity; the molecular biology method has high specificity, but is limited by the degree of preservation of the genetic material in the sample. For sea dragon medicinal wine, processed products, extracts, insect-infested and moldy samples, and samples mixed with multiple individuals, it is difficult to extract DNA and cannot be successfully experimented. In recent years, more and more methods and standards use characteristic peptides to identify or quantitatively study natural drugs. The proteins contained in each species have differences in individual amino acids. Appropriate proteases can be used to extract the difference sites as polypeptide segments with identification significance, namely characteristic peptides. The detection of characteristic peptides generally uses mass spectrometry, without considering protein activity, and the preparation method is simple and the peptide properties are relatively stable. The existing methods for identifying sea dragons have many limiting factors and cannot be applied to sea dragon powder, sea dragon medicinal wine, processed products, extracts and other derivative products. The characteristic peptides in sea dragon proteins have the potential to be used as indicator components for sea dragon species identification. At present, the protein sequence information related to Baojia lance-mouthed sea dragon is not included in various databases, and differential peptides cannot be obtained through protein sequence comparison. Therefore, there is no record of the characteristic peptides of Baojia lance-mouthed sea dragon. Summary of the invention

[0005] The purpose of the present invention is to provide a characteristic polypeptide of Hailong baojia, its application and a method for identifying Hailong baojia. The characteristic polypeptide provided by the present invention has excellent specificity and stability for Hailong baojia, strong specificity, can be used for the identification of Hailong medicinal materials, specifically the identification of Hailong baojia, and has good application prospects.

[0006] The present invention provides a characteristic polypeptide of Hailong baojia, and the amino acid sequence of the characteristic polypeptide is shown in SEQ ID NO.1.

[0007] The present invention also provides the application of the characteristic polypeptide described in the above technical solution in identifying the Baojialonglong.

[0008] Preferably, the identification is performed using mass spectrometry, and the detected ion pairs include: a quantitative ion with a mass-to-charge ratio of m / z 728.5→570.3 for SEQ ID NO.1, and a qualitative ion with a mass-to-charge ratio of m / z 728.5→1116.6.

[0009] The present invention also provides a method for identifying the Baojia Hailong, using the characteristic polypeptide shown in SEQ ID NO.1 as a reference substance.

[0010] Furthermore, the present invention also provides a method for identifying the Baojia lance-nosed sea dragon based on the characteristic polypeptide described in the above technical solution, comprising the following steps:

[0011] (1) Preparation of the test solution: The pretreated sea dragon extract or the pretreated sea dragon powder extract is mixed with trypsin to obtain a mixed solution, the mixed solution is mixed with an aqueous solution of ammonium bicarbonate, and the mixture is filtered after enzymatic hydrolysis to obtain a test solution;

[0012] (2) Preparation of reference solution: using the characteristic peptide of the sea dragon described in the above technical solution as a reference substance, adding water to dissolve it to obtain a reference solution;

[0013] (3) Detection and analysis: Detection and analysis were performed using triple quadrupole mass spectrometry;

[0014] There is no time sequence limitation between the steps (1) and (2).

[0015] Preferably, in step (1), the method for preparing the pretreated sea dragon extract comprises the following steps: crushing the sea dragon, mixing it with water and decocting it three times, the decocting time being 4 hours, 3 hours and 2 hours respectively, combining the decoctions, and diluting them with an aqueous solution of ammonium bicarbonate to obtain the pretreated sea dragon extract;

[0016] The preparation method of the pretreated sea dragon powder extract comprises the following steps: mixing sea dragon powder with water and decocting three times, the decocting time is 4 hours, 3 hours and 2 hours respectively, combining the decoctions, and diluting with an ammonium bicarbonate aqueous solution to obtain the pretreated sea dragon powder extract;

[0017] Preferably, the trypsin is added in the form of a trypsin aqueous solution, and the mass concentration of the trypsin in the trypsin aqueous solution is 1 mg / ml.

[0018] Preferably, the mass percentage of ammonium bicarbonate in the ammonium bicarbonate aqueous solution is 1%.

[0019] Preferably, the enzymatic hydrolysis condition is 37° C. for 2 h.

[0020] Preferably, the triple quadrupole mass spectrometry method uses 0.1% formic acid acetonitrile as mobile phase A and 0.1% formic acid solution as mobile phase B, and performs gradient elution: 0-3 min, 5% A→8% A; the flow rate is 0.5 ml per minute.

[0021] Preferably, the triple quadrupole mass spectrometry uses a mass spectrometer detector in electrospray positive ion mode to perform multiple reaction monitoring, and SEQ ID NO.1 selects a quantitative ion with a mass-to-charge ratio of m / z728.5→570.3 and a qualitative ion with a mass-to-charge ratio of m / z728.5→1116.6 as the detection ion pair.

[0022] The present invention provides a characteristic polypeptide of the Baojia lance-mouthed sea dragon. The characteristic polypeptide provided by the present invention can be used as an indicator component for identifying the Baojia lance-mouthed sea dragon, and can be distinguished from sea dragons of other species, laying a foundation for developing an identification method for the Baojia lance-mouthed sea dragon and related products. Specifically, the present invention has the following beneficial effects:

[0023] (1) The present invention extracts the differential polypeptide segments in the protein sequence and uses mass spectrometry for detection. The operation is simple and fast, the measurement results are stable, and it is not affected by the morphology and processing of the sea dragon sample. It fills the gap in the species identification of the Baojia lance-mouthed sea dragon and the detection method of the Baojia lance-mouthed sea dragon in sea dragon products.

[0024] (2) The characteristic peptides and detection methods provided by the present invention provide a reference for finding characteristic peptides in closely related species that are lacking in the database;

[0025] (3) The characteristic polypeptide provided by the present invention has excellent specificity and stability for the Baojia lance-wen sea dragon, has strong specificity, can be used for the identification of Baojia lance-wen sea dragon medicinal materials, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The attached figure is a specific map of the characteristic peptide SEQ ID NO.1 of the Baojialonglong provided by the present invention;

[0027] in: Figure 1 : SEQ ID NO.1 characteristic peptide control; Figure 2 : Baojia snatches the sea dragon sample for kissing; Figure 3 : Diao Hailong sample; Figure 4 : Sample of Pseudohalodon; Figure 5 : Sample of Polyspinus multispinus; Figure 6 : Sample of Ge's sea bracelet fish; Figure 7 : Sample of Roughnose Sea Dragon; Figure 8 : Long Thick-nosed Sea Dragon sample; Fig. 9 : Sample of Schweini's Sea Dragon. DETAILED DESCRIPTION

[0028] The present invention provides a characteristic polypeptide of Baojia lance-snout sea dragon, the amino acid sequence of which is shown in SEQ ID NO.1: ANGP-(HYP)-GATGF-(HYP)-GAPGR, and the present invention provides a peptide segment with characteristic identification significance of Baojia lance-snout sea dragon. The present invention hydrolyzes water extracts of different species of sea dragons such as Baojia lance-snout sea dragon, pseudo-sea dragon, diao sea dragon, and shu's sea dragon with trypsin, and then uses nano-liquid chromatography-high-resolution mass spectrometry to analyze and obtain a mass spectrum of the full peptide segment of sea dragon protein; the mass spectrum data is subjected to chemometric analysis, and then a triple quadrupole mass spectrometer is used to verify the specificity of the experimental results; finally, a characteristic ion pair is provided, corresponding to a characteristic peptide segment, and the specificity verification result is good.

[0029] The present invention also provides the application of the characteristic polypeptide described in the above technical solution in identifying the Baojialonglong.

[0030] In the present invention, the identification is performed using mass spectrometry, and the detected ion pairs include: a quantitative ion with a mass-to-charge ratio of m / z 728.5→570.3 for SEQ ID NO.1, and a qualitative ion with a mass-to-charge ratio of m / z 728.5→1116.6.

[0031] The present invention also provides a method for identifying the Baojialonglong based on the characteristic polypeptide described in the above technical solution, comprising the following steps:

[0032] (1) mixing the pretreated sea dragon extract or the pretreated sea dragon powder extract with trypsin to obtain a mixed solution, mixing the mixed solution with an aqueous solution of ammonium bicarbonate, filtering after enzymatic hydrolysis, and taking the filtrate to obtain a test solution;

[0033] (2) using the characteristic peptide of the sea dragon described in the above technical solution as a reference substance, adding an aqueous solution of ammonium bicarbonate to dissolve it to obtain a reference substance solution;

[0034] (3) Detection and analysis using triple quadrupole mass spectrometry;

[0035] There is no time sequence limitation between the steps (1) and (2).

[0036] The method of the present invention extracts differential polypeptide segments from the protein sequence and uses mass spectrometry for detection. The operation is simple and fast, the measurement results are stable, and the method is not affected by the morphology and processing of the sea dragon sample. It fills the gaps in the species identification of the Baojia lance-billed sea dragon and the detection method of the Baojia lance-billed sea dragon in sea dragon products, and provides a reference for the search for characteristic peptide segments in closely related species that are lacking in the database.

[0037] The present invention mixes the pretreated sea dragon extract or the pretreated sea dragon powder extract with trypsin respectively to obtain a mixed solution, mixes the mixed solution with an aqueous solution of ammonium bicarbonate, filters after enzymolysis, and takes the filtrate to obtain a test solution. In the present invention, the preparation method of the pretreated sea dragon extract preferably includes the following steps: crushing the sea dragon, mixing it with water and decocting it three times, the decocting time is 4h, 3h and 2h respectively, combining the decoctions, and diluting it with an aqueous solution of ammonium bicarbonate to obtain a pretreated sea dragon extract. The present invention preferably takes 100g of crushed sea dragon, and the decoction preferably totals 500mL. In the present invention, the dilution is preferably diluted 10 times. Specifically, the present invention preferably takes 5mL of the decoction, places it in a 50mL volumetric flask, dilutes it to the scale with an aqueous solution of ammonium bicarbonate, shakes well, and obtains a pretreated sea dragon extract. In the present invention, the preparation method of the pretreated sea dragon powder extract preferably comprises the following steps: mixing sea dragon powder with water and decocting three times, the decocting time is 4h, 3h and 2h respectively, combining the decoctions to obtain the pretreated sea dragon powder extract. The present invention preferably takes 1g of sea dragon powder, and the decoction is preferably 50mL in total. Specifically, the present invention preferably takes 5mL of the decoction, places it in a 50mL volumetric flask, dilutes it to the scale with an aqueous solution of ammonium bicarbonate, shakes well, and obtains a pretreated sea dragon powder extract. After obtaining the pretreated sea dragon extract and the pretreated sea dragon powder extract, the present invention preferably measures 1 to 5mL of sea dragon extract or sea dragon powder extract, preferably adds 100μL of trypsin solution, shakes well, adds an aqueous solution of ammonium bicarbonate to 50mL, shakes well, and performs enzymolysis after sealing. In the present invention, the enzymolysis conditions are preferably 37℃ enzymolysis for 2h. After enzymolysis, the present invention preferably cools, more preferably cools to room temperature, and the temperature range of room temperature in the present invention is preferably 10 to 30℃. After cooling, the present invention performs filtering, and takes the filtrate as the test solution.

[0038] In the present invention, the trypsin is added in the form of a trypsin aqueous solution, and the mass concentration of the trypsin in the trypsin aqueous solution is 1 mg / ml. In the present invention, the mass percentage of ammonium bicarbonate in the ammonium bicarbonate aqueous solution is 1%.

[0039] The present invention uses the characteristic peptide of the Baojia lance-wen sea dragon described in the above technical solution as a reference substance, adds water to dissolve it, and obtains a reference substance solution. The present invention has no special limitation on the source of the characteristic peptide of the Baojia lance-wen sea dragon, and an artificial synthesis method can be used. For example, the characteristic peptide of the Baojia lance-wen sea dragon in the present invention is obtained by entrusting Nanjing Yuanpeptide Biotechnology Co., Ltd. to synthesize it according to the specified amino acid sequence.

[0040] The present invention uses triple quadrupole mass spectrometry for detection and analysis. In the present invention, the triple quadrupole mass spectrometry preferably uses 0.1% formic acid acetonitrile as mobile phase A and 0.1% formic acid solution as mobile phase B, and performs gradient elution: 0-3min, 5%A→8%A; the flow rate is 0.5ml per minute. In the present invention, the triple quadrupole mass spectrometry preferably uses a mass spectrometer detector, electrospray positive ion mode, and multiple reaction monitoring. SEQ ID NO.1 selects a quantitative ion with a mass-to-charge ratio of m / z 728.5→570.3 and a qualitative ion with a mass-to-charge ratio of m / z 728.5→1116.6.

[0041] The following is a further detailed introduction to the characteristic polypeptide of the present invention, its application and the method for identifying the present invention in conjunction with specific examples. The technical solutions of the present invention include but are not limited to the following examples.

[0042] Triple quadrupole mass spectrometry conditions

[0043] 0.1% formic acid acetonitrile was used as mobile phase A, and 0.1% formic acid solution was used as mobile phase B, and gradient elution (0-3 min, 5% A→8% A) was performed; the flow rate was 0.5 ml per minute. A mass spectrometer was used in electrospray positive ion mode (ESI+) to perform multiple reaction monitoring (MRM), and SEQ ID NO.1 selected a quantitative ion with a mass-to-charge ratio of m / z 728.5→570.3 and a qualitative ion with a mass-to-charge ratio of m / z 728.5→1116.6.

[0044] Example 1: Search for characteristic peptides of Hailong baojia

[0045] (1) Preparation of test solution

[0046] Sea dragon water extract: Take 100g of different species of sea dragon samples, grind them, put them in a conical flask, add water and boil them three times, for 4h, 3h, and 2h respectively, combine the decoctions to make a total of 500mL, boil slightly and concentrate until the liquid is viscous, transfer to a silica gel bowl, and dry them in a 60℃ electric constant temperature blast drying oven until solid, to obtain the sea dragon water extract;

[0047] Test solution: weigh 0.1 g of the water extract of the sea dragon, add 50 ml of 1% ammonium bicarbonate aqueous solution, ultrasonically treat for 30 min to dissolve, filter with a microporous filter membrane, take 100 μL of the filtrate, add 10 μL of 1 mg / ml trypsin aqueous solution, enzymatically hydrolyze at 37°C for 2 h, take out and cool to room temperature.

[0048] (2) Selection of characteristic ions and sequence inference

[0049] After the sample solution was analyzed by nano-liquid chromatography-high-resolution mass spectrometry, the mass spectrometry data was imported into PEAKS 8.5 software for de novo sequencing and sequence prediction of all peptides of the sample, and the results were analyzed. From the analysis results, peptides that were only detected in the Baojia lance-snout sea dragon and almost not detected in other species of sea dragons were selected as parent ions, and their secondary spectra were analyzed one by one to select daughter ions with better responses. The mass spectrometry ion pair information of Baojia lance-snout sea dragon was finally selected as follows: m / z 728.5 (double charge) → 570.3, 1116.6. Through further analysis, it was inferred that the characteristic peptide sequence of Baojia lance-snout sea dragon corresponding to the ion was ANGP-(HYP)-GATGF-(HYP)-GAPGR, where HYP is hydroxyproline.

[0050] According to the inferred amino acid sequence, a characteristic peptide reference substance of the Baojia Longyan Dragon was commissioned to be synthesized. The reference substance and the Baojia Longyan Dragon test sample solution were tested simultaneously. The retention time and secondary mass spectrometry information of the two were consistent, thus confirming the correctness of the sequence.

[0051] Example 2: Study on the specificity of the characteristic peptide of Hailong baojia

[0052] The water extracts of all species of sea dragons were tested using triple quadrupole mass spectrometry. The results showed that the three batches of Baojia lance-mouthed sea dragon species all had Baojia lance-mouthed sea dragon characteristic peptide chromatographic peaks, while other multi-spined sea dragons, pseudo-sea dragons, schumeri sea dragons, diao sea dragons, long thick-snout sea dragons, and Ge's sea dragons did not have corresponding chromatographic peaks. See attached Figure 1-9 The results showed that the characteristic peptide was unique to T. baojiaensis and could be used as an indicator component to identify T. baojiaensis.

[0053] Example 3: Application of the characteristic peptide of Baojialonghailong in sea dragon extract and sea dragon powder

[0054] (1) Sample preparation

[0055] Preparation of the test solution of sea dragon extract: Take 100g of the test sea dragon, crush it, put it in a conical flask, add water and boil it three times, for 4h, 3h, and 2h respectively, and combine the decoctions to make a total of 500mL; take 5ml of sea dragon extract, put it in a 50ml volumetric flask, add 1% ammonium bicarbonate aqueous solution to dilute to the scale, shake well, measure 1ml to a 5ml volumetric flask, add 100μl of 1mg / ml trypsin aqueous solution, shake well, then add 1% ammonium bicarbonate aqueous solution to make up to the scale, shake well, seal it, and hydrolyze it at 37℃ for 2h, cool it, filter it, and take the filtrate as the test solution.

[0056] Preparation of the test solution of sea dragon powder extract: Take 1 g of the test sea dragon, grind it, put it in a conical flask, add water and boil it three times, for 4h, 3h, and 2h respectively, combine the decoctions, a total of 50mL, measure 1ml into a 5ml volumetric flask, and prepare according to the above method starting from "add 100μl of 1mg / ml trypsin aqueous solution".

[0057] Baojia lance-wen sea dragon characteristic peptide reference substance solution: weigh 10 mg of Baojia lance-wen sea dragon characteristic peptide reference substance SEQ ID NO.1 respectively, put it into a 50 ml volumetric flask, add water to dissolve and dilute to the scale to obtain.

[0058] (2) The determination was performed using triple quadrupole mass spectrometry conditions.

[0059] (3) Sample measurement

[0060] Ten batches of Hailong Baojia extract samples and eight batches of Hailong Baojia powder samples were collected and tested, and the results showed that the characteristic peptide of Hailong Baojia was detected in all of them, indicating that this characteristic peptide can be used to identify Hailong Baojia components in Hailong products.

[0061] Example 4: Ion information with identification potential

[0062] Seven ions with identification potential were screened out from 330 candidate ions. After verification by triple quadrupole mass spectrometry, it was found that not all ions could be used to identify the components of T. baojiaensis. Some ions were not specific enough for T. baojiaensis. Only the ion with a mass-to-nuclear ratio of 728.5 had good specificity. The specific results are shown in Table 1.

[0063] Table 1 Specificity verification results

[0064]

[0065] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A polypeptide characteristic of the sea dragon, characterized in that: The amino acid sequence of the characteristic polypeptide is shown in SEQ ID NO.

1.

2. Use of the characteristic polypeptide according to claim 1 in identifying the Baojialonglonglong.

3. The use according to claim 2, characterized in that: The identification is performed using mass spectrometry, and the ion pairs detected for SEQ ID NO.1 include: a quantitative ion with a mass-to-charge ratio of m / z 728.5→570.3, and a qualitative ion with a mass-to-charge ratio of m / z 728.5→1116.

6.

4. A method for identifying the Baojia sea dragon, characterized in that: The following steps are involved: (1) Preparation of the test solution: the pretreated sea dragon extract or the pretreated sea dragon powder extract is mixed with trypsin to obtain a mixed solution, the mixed solution is mixed with an aqueous solution of ammonium bicarbonate, and the mixture is filtered after enzymatic hydrolysis to obtain the test solution; (2) Preparation of reference solution: using the characteristic peptide of the sea dragon of Baojia as claimed in claim 1 as a reference substance, adding water to dissolve it to obtain a reference solution; (3) Detection and analysis: Detection and analysis are performed using triple quadrupole mass spectrometry; There is no time sequence limitation between the steps (1) and (2); The method for preparing the pretreated sea dragon extract comprises the following steps: crushing the sea dragon, mixing it with water and decocting it three times, the decocting time being 4 hours, 3 hours and 2 hours respectively, combining the decoctions, and diluting them with an aqueous solution of ammonium bicarbonate to obtain the pretreated sea dragon extract; The preparation process of the pretreated sea dragon powder filtrate comprises the following steps: mixing sea dragon powder with water, decocting for 1 hour, filtering, taking the filtrate, and obtaining the pretreated sea dragon powder filtrate; The trypsin is added in the form of a trypsin aqueous solution, and the mass concentration of the trypsin in the trypsin aqueous solution is 1 mg / ml; The mass percentage of ammonium bicarbonate in the ammonium bicarbonate aqueous solution is 1%; the enzymatic hydrolysis condition is 37° C. for 2 hours; The triple quadrupole mass spectrometry method uses 0.1% formic acid acetonitrile as mobile phase A and 0.1% formic acid solution as mobile phase B, and performs gradient elution: 0-3 min, 5% A→8% A; the flow rate is 0.5 ml per minute; The triple quadrupole mass spectrometry method uses a mass spectrometer detector in electrospray positive ion mode to perform multiple reaction monitoring. SEQ ID NO.1 selects a quantitative ion with a mass-to-charge ratio of m / z 728.5→570.3 and a qualitative ion with a mass-to-charge ratio of m / z 728.5→1116.6 as the detection ion pair.

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